Structural basis for self-discrimination by neoantigen-specific TCRs

T cell receptors (TCR) are pivotal in mediating tumour cell cytolysis via recognition of mutation-derived tumour neoantigens (neoAgs) presented by major histocompatibility class-I (MHC-I). Understanding the factors governing the emergence of neoAg from somatic mutations is a major focus of current research. However, the structural and cellular determinants controlling TCR recognition of neoAgs remain poorly understood. This study describes the multi-level analysis of a model neoAg from the B16F10 murine melanoma, H2-Db/Hsf2 p.K72N68-76, as well as its cognate TCR 47BE7. Through cellular, molecular and structural studies we demonstrate that the p.K72N mutation enhances H2-Db binding, thereby improving cell surface presentation and stabilizing the TCR 47BE7 epitope. Furthermore, TCR 47BE7 exhibited high functional avidity and selectivity, attributable to a broad, stringent, binding interface enabling recognition of native B16F10 despite low antigen density. Our findings provide insight into the generation of anchor-residue modified neoAg, and emphasize the value of molecular and structural investigations of neoAg in diverse MHC-I contexts for advancing the understanding of neoAg immunogenicity.

High functional avidity/structural affinity has emerged as a recurrent feature of neoAg-reactive TCRs 26 and may be necessary to recognise tumour cells naturally selected for low target antigen surface density.In early examples, this has been shown to derive from TCR recognition of structural differences between mutation-derived neoAg peptide and the corresponding wild-type (WT) peptide [27][28][29] , but the broader generalizability of these findings remains unknown.Many other core questions remain unanswered, such as why nonsynonymous mutations are rarely recognised by TCRs; and how some neoAg-reactive TCRs selectively recognise mutated peptides and do not cross-react with the corresponding wild-type peptides, whereas others exhibit significant cross-reactivity [27][28][29][30] .Structure-guided mechanistic answers to these questions might enable the prediction of neoAg-reactive TCR activity as well as potential toxicities resulting from cross-reactivity, potentially enabling the rapid translation of safe and effective neoAg-reactive TCRs into the clinic.
To systematically address these questions pertaining to neoAgreactive TCRs, we employed the B16F10 murine melanoma cell line, an orthotopic implantable tumour model syngeneic to C57BL/6 mice that exhibits limited spontaneous immunogenicity and is refractory to multiple types of immunotherapy, including checkpoint blockade 31 .We reasoned that neoAgs identified in this model would approximate neoAgs observed in advanced human cancers more closely than exogenous model antigens such as ovalbumin 32 , thereby improving the biological relevance of our findings.Furthermore, there are several well-known conventional tumour-associated antigens (gp100, Trp2, Tyrp1) relevant in the B16F10 model which can serve as comparators for functional and structural studies.These tumour-associated antigens (TAAs) often do not elicit a robust or exclusively tumour-specific endogenous anti-tumour immune response and corresponding tumour growth control 33,34 , underscoring the need to identify and study neoAgs in the B16F10 model.
Here we perform whole exome and transcriptomic sequencing of B16F10 and characterise a subset of expressed non-synonymous mutations via in vivo validation.We immunise mice with synthetic peptides corresponding to selected mutations and characterise the vaccine-induced CD8 + T cell response to seven neoAgs from over 50 predicted neoAgs.We isolate, clone, and perform functional analyses of cognate TCRs recognizing each neoAg.Among them, only the TCR targeting H2-D b -restricted Hsf2 p.K72N ('p' indicating peptide residue) confers specific recognition of the B16F10 cells in vitro and demonstrates anti-tumour effect in vivo, albeit dependent upon sufficient tumour expression of neoAg Hsf2 p.K72N .Finally, using biochemical and cellular assays in combination with high-resolution crystal structures of the neoAg Hsf2 p.K72N-H2-D b complex, with and without a corresponding reactive TCR, we determine the structural requirements for TCR antigen recognition and selectivity.We observe that Hsf2 p.K72N is discriminated by both the MHC and cognate TCR from the WT Hsf2.We determine that Hsf2 p.K72N is a group II neoAg with a mutation at an anchor residue.Group II neoAg are typically minimally cross-reactive with their corresponding WT peptides due to discrimination at the MHC level and thereby may resemble non-self epitopes generated in the course of viral or bacterial infections 35 .

Identification of neoantigens in B16F10 melanoma
To identify B16F10 neoAgs, we performed paired exome sequencing of cultured B16F10 murine melanoma tumour cells and reference C57BL/6 splenocytes, as well as bulk RNASeq analysis of resected B16F10 tumours (Fig. 1a).Variant expression was quantified by local assembly and allele-specific quantification of mutated and reference transcripts.The peptide-MHC-I binding prediction tool NetMHCpan (v.4.1) was then used to identify candidate neoAg for further study, in accordance with published methods 36,37 .We then performed murine immunization studies using SNAPvax™, a peptide-based vaccine that is conjugated to an adjuvant small molecule imidazoquinone-based Tolllike Receptor 7/8 agonist (TLR7/8a) and contains charge-modifying groups to accommodate a wide variety of peptide chemistries; this cancer vaccine platform has yielded robust anti-tumour T cell immunity enabled by enhancements in dendritic cell recruitment and antigen uptake, which in turn reduced tumour growth in multiple models 38,39 .For these initial immunization studies, we developed SNAPvax™ formulations incorporating twelve distinct 25mer ("long") neoantigenic peptides (Supplementary Table 1).To minimise the risk of antigenic competition, screening immunization was performed individually with one peptide antigen specificity, for each of the 12 tested neoAg, and three control non-mutated tumour-associated antigens (TAA) [40][41][42] .We observed both vaccine-elicited neoAg-specific CD4 + and CD8 + T cells amongst splenocytes for 4 and 7 neoAgs, respectively, of nearly 50 long peptide-derived MHC-I-restricted neoAg peptides surveyed (Fig. 1b, c, Supplementary Figs. 1, 2), as defined by robust IFNγ production by T cells in response to stimulation with neoantigenic peptides and, for the CD8 + T cell compartment specifically, tetramer staining of T cells with MHC-I tetramers.To determine the minimal peptide epitope for the 7 MHC-I-restricted neoAg hits, we immunised mice with various minimal epitopes derived from the long peptide vaccine formulations and assessed neoAgreactive T cell yield via tetramer staining, akin to that performed in Fig. 1b (Fig. 1d).A summary of these immunogenic MHC-I-restricted epitopes, as well as four non-mutated previously characterised tumour antigens [40][41][42] , and their predicted binding affinities of wild type (WT) versus mutant (MT) peptide to MHC is shown; these predictions revealed that pMHCs in our system span the spectrum of mutant peptide-MHC affinity and specificity for mutant peptide (ratio of mutant to wild type affinity for MHC) (Fig. 1e, f).Altogether, we observed that predicted neoAgs are indeed immunogenic in vivo and determined the minimal epitopes that elicit T cell immunity for seven neoAgs, enabling further characterization.

Stabilisation of pMHC by mutant-specific anchor residue
Previous studies have shown that the position of the mutated amino acid with respect to peptide length can be used to organise neoAgs into two principal groups 43,44 .Namely, neoAgs in which the mutated amino acid side-chain is solvent facing and may form inter-molecular bonds with incoming TCR directly (group I); versus neoAgs in which the mutated amino acid side-chain is not solvent facing, and instead buried within and interacts predominately with the MHC-I binding pocket (group II).
Hsf2 p.K72N 68-76 is a H2-D b -restricted mutated peptide ( 68 YGFRNVVHI 76 ) derived from Heat shock factor 2 (Hsf2, Uniprot: P38533).The underlying point mutation results in substitution of a basic lysine (Lys/K) residue at position 5 of WT Hsf2 68-76 (pK 5 ; 'p' indicating peptide residue, with the number designating the position of the residue in the peptide starting from the N-terminus) for a polar noncharged residue asparagine (Asn/N (pN 5 )).In silico binding analysis with NetMHCPan4.1 predicted that both Hsf2 p.K72N 68-76 [0.007%Rank] and WT Hsf2 68-76 [0.29%Rank] can bind H2-D b , albeit with a significantly different affinity (Fig. 1f).To confirm the observed difference in binding affinity we performed cell-based RMA-S in vitro binding assays, which showed half-maximal stabilisation of cell surface H2-D b (EC 50 ) by Hsf2 p.K72N 68-76 at 4.985 nM that was comparable to that of control agonist peptide LCMV gp 33-41 4.38 nM, and significantly lower than that of Hsf2 68-76 883 nM (Fig. 5a).Based on this observation we hypothesised that H2-D b /Hsf2 p.K72N is a murine prototype group II neoAg 35 , in which immunogenicity is at least in part derived from improved binding of the MT peptide with H2-D b , likely secondary to the mutated residue pN 5 .Furthermore, given its high predicted MHC-I binding affinity for MT peptide, with a large differential in binding affinity between the MT and WT peptide, we hypothesised that detailed structural characterization of the H2-D b /Hsf2 p.K72N 68-76 complex might elucidate principles governing the immunogenicity of group II neoAgs.
Analysis of published H2-D b crystal structures demonstrated a conserved peptide binding mode mediated by hydrophobic interactions between conserved residues lining the H2-D b A-B-D and F-pockets and peptide N-/C-terminal anchor residues 45 .Additionally, H2-D b bound peptides characteristically form polar interactions within the MHC-I C-pocket, mediated by bi-directional hydrogen bonds between H2-D b Q 97 and pN 5 .The hydrogen bond mediated by H2-D b Q 97 leads to its biased presentation of peptides with pN 5 .Lastly, H2-D b is defined by a conserved hydrophobic bridge formed by the side chains of W 73 (αhelix), W 147, and Y 156 (α-helix) that runs perpendicular to the binding cleft and imparts an arched solvent-accessible conformation to residues in p6-p8 of H2-D b bound peptides which is absent from H2-K b bound peptides 45 .We hypothesised that Hsf2 p.K72N 68-76 , but not WT Hsf2 68-76 , satisfied the pN 5 requirement imposed by H2-D b , and that the p6-p8 residues would form a solvent-exposed ridge accessible to the incoming TCR.To validate this notion, we produced the soluble hβ2M/ H2-D b /Hsf2 p.K72N 68-76 (YGFRNVVHI) pMHC complex, then crystallised and solved its crystal structure to a resolution of 1.74 Å (Fig. 5b) 46,47 .In the structure, we observed a typical pMHC fold, in which the peptide binding is mainly supported by interactions between the H2-D b residues at the A, B, D, C and F-pockets and the buried peptide residues pY 1 -pF 3 and pI 9 , respectively-all of these residues are conserved between both MT/WT peptides (Supplementary Fig. 7a, b).The A-pocket is occupied by the pY 1 side chain, which is solvent facing but remains mostly buried, with the terminal amine engaged in hydrogen bonds with H2-D b Y 7 , Y 59, and Y 171 .The B-and D-pockets are occupied by pG 2 and pF 3 , both of which are also buried within the H2-D b binding cleft, and pR 4 , which is solvent exposed (Fig. 5c-e).However, the respective residues exhibit poor compatibility with B-and D-pockets, which are filled with water, possibly weakening binding and increasing thermal flexibility of the pY 1 -pR 4 segment.This notion is supported by the elevated B-factor values associated with the epitope's N-terminus and the adjacent helices in the binary complex structure (Fig. 5c).The hydrophobic F-pocket is occupied by anchor pI 9 , which is stabilised by van der Waals (VDW) interactions and the H-bonds between its terminal carboxyl group and the H2-D b N 80 and K 146 side chains.
The pN 5 side chain is hidden inside the C-pocket and its primary amide is engaged in a hydrogen bond network with the H2-D b Q 97 side chain, functioning as a mutant-specific anchor residue (Fig. 4d, e).Such arrangement is typical between H2-D b and pN 5epitopes and is often observed with non-pN 5 -peptides (Supplementary Fig. 7c-e), with some exceptions discussed below.We show the superimposition of the H2-D b structures in complex with Hsf2 NeoAg (PDB 7N9J) or NP-N3D (PDB 4L8C) flu epitope, which share highly similar conformations, despite having only one (pN 5 ) (without IFNγ)' to serve as a negative control.'WT (Ef1a vector)' indicates that B16F10 was transduced with an "empty antigen" Ef1a lentiviral construct, meanwhile 'OE (Ef1a vector)' indicates that B16F10 was transduced (for antigen overexpression [OE]) with the appropriate antigen matching the TCR indicated.identical residue (Supplementary Fig. 7) 48 .The presence of pN 5 usually correlates with increased affinity between peptide and H2-D b .For instance, substitution of pN 5 with another residue usually results in a significant affinity drop between such peptides and H2-D b which correlates with our data presented in Fig. 5a 49 .
The C-terminal peptide side chains-pR 4 and pV 6 -pV 7 -pH 8 -are solvent-exposed and do not directly contribute to MHC-I binding.Residues pV 6 and pV 7 form an arch over the W 73 -W 147 -Y 156 bridge that projects towards A 152 of the α 2 -helix, with pH 8 projecting back towards the same helix.This type of arrangement is common for H2-D b and mostly observed independent of the peptide amino acid sequence (Supplementary Fig. 7c-e) with a possible exception of the peptides with nonpolar or aromatic side chains at positions p 5 and p 6 50 .Analysis of crystallographic B-factors revealed asymmetry in the distribution of peptide all-atom flexibility with N-terminal residues proximal to the pN 5 anchor exhibiting increased average B-factor values relative to distal C-terminal residues, pY 1 -pR 4 [39.05 ± 4.97] versus pN 5 -pI 9 [27.64 ± 1.56] (Fig. 5c).Overall, this arrangement suggests H2-D b /Hsf2 p.K72N 68-76 is stabilised primarily at the C-terminus, via the contribution from conserved pI 9 anchor as well as the pN 5 anchor.Additionally, the increased thermal rigidity of the C-terminal region and elevated solvent exposure altogether suggest that the pY 1 -pR 4 epitope segment may be preferentially targeted by TCRs 45 .
In summary, the H2-D b /Hsf2 p.K72N 68-76 crystal structure revealed a typical H2-D b peptide binding mode mediated by hydrophobic interactions between residues lining the H2-D b A-B-D and F-pockets and peptide N-/C-terminal anchor residues 45 .Additionally, the H2-D b bound epitope with pN 5 supports bi-directional hydrogen bonds between the H2-D b Q 97 and peptide anchor pN 5 side chains.Thus, the K72N mutation in Hsf2 gene has produced a prototypical group II neoAg with high affinity for H2-D b .In contrast, the WT Hsf2 peptide had low affinity toward H2-D b .

TCR-47BE7 preference for Hsf2 p.K72N 68-76
Having identified a plausible physical mechanism for enhanced binding of the neoAg Hsf2 p.K72N 68-76 to H2-D b , we sought to better understand the preference of the 47BE7 TCR for the MT peptide K72N 68-76 observed in earlier functional studies (Fig. 2c, d).Selectivity for the MT peptide is an important characteristic of neoAgreactive TCRs that could provide effective tumour growth control with minimal off-target effects relative to that observed upon treatment with TCRs responsive to tumour-associated antigens 35 .In cell-based cytokine production assays, we found TCR 47BE7 to be approximately 1.55×10 6 fold more sensitive to the MT peptide (EC 50 , 5.6pM) relative to the WT peptide (EC 50 , 8.7 µM) (Fig. 2d).This difference was several orders of magnitude larger than the 175-fold  difference observed in the RMA-S MHC-I stabilisation assay (Fig. 5a).Moreover, in response to saturating peptide concentrations, we observed significantly greater cytokine production on a per-cell basis when exposed to MT versus the WT peptide (Fig. 2c), suggesting that the WT peptide is a weak agonist.Altogether, these data indicated that the MT and WT Hsf2 peptides are discriminated by TCR 47BE7 as well as by H2-D b .
To further characterise the biochemical basis for antigen discrimination by TCR 47BE7 we generated a positional scanning peptide library wherein each position within Hsf2 p.K72N 68-76 was replaced with each of the remaining 19 protein-coding amino acids, and then assessed cytokine production by 47BE7-expressing tgTCR CD8 + T cells (Fig. 5f, Supplementary Fig. 8).The majority of sidechain substitutions of the non-core anchor residues pY 1 -pG 2 and pI 9 were generally tolerated.A limited set of conservative, primarily aromatic substitutions was tolerated at p3, rendering these residues non-essential for TCR 47BE7 binding.Conversely, the vast majority of side-chain substitutions within the core epitope pR 4 -[X]-pV 6 -pV 7 -pH 8 abolished TCR recognition and therefore were considered essential for TCR 47BE7 binding.Exceptions included biochemically conservative substitutions for the polar-basic residue pR 4 (replaced with Q, T) and non-polar residues pV 6 (replaced with I, Y) and pV 7 (replaced with L, M).Notably, TCR 47BE7 tolerated multiple sidechain substitutions at p5 .This observation could be explained by both the lack of significant interfacial contacts between TCR 47BE7 and the side chain of pN 5 , as well as that other residues at p5 allow the same or similar conformation of the core neoepitope (Supplementary Fig. 7a).Consistent with this, comparison of available H2-D b structures demonstrates that alternative p5 residues, including glycine, alanine, aspartate, histidine and methionine, adopt anchor conformations similar to that of asparagine (Supplementary Fig. 7e).In our studies, the exceptions from the above rule were the two substitutions at p5, lysine and leucine, respectively.The corresponding mutant peptides were unable to produce detectable activation of the TCR 47BE7-expressing T cells in the given conditions (Fig. 4f).Because strong peptide-MHC interaction is necessary for TCR binding and T cell activation [51][52][53] , we performed in silico binding affinity analysis of all position 5 substituted peptides using NetMHCPan4.1.We observed a weak but significant direct correlation between predicted affinity to MHC-I and the TCR 47BE7 activation (Supplementary Fig. 7f).Notably, the WT Hsf2 68-66 (YGFRKVVHI) was an outlier with no T cell activation detected, suggesting that peptide-MHC binding affinity alone was not sufficient to explain the selectivity of TCR-47BE7.
Using the peptide scan data presented in Fig. 4f, we created a ProSite search pattern - 54 and utilised this pattern to identify sequences in mouse proteome potentially cross-reactive with TCR 47BE7.The initial search produced a total of 107 hits (Supplementary Data 1).Based on affinity estimates by NetMHCPan4.1,only one peptide satisfied the epitope selection criteria (NVFRNILHV, Uniprot ID Q9D3N2, with an estimated K D value below 1 μM).However, comparison with the mouse immunopeptidome ruled out this epitope, as it was not detected in complex with H2-D b in published mouse tissue analysis databases 55 .
In summary, the peptide scan analysis combined with the crystal structure of the binary complex suggest that the core pattern R 4 -X-V 6 -V 7 -H 8 of the Hsf2 p.K72N 68-76 epitope is selectively recognised by TCR 47BE7.The lack of significant similarity between the R 4 -X-V 6 -V 7 -H 8 pattern and the mouse proteome and immunopeptidome renders the possibility of significant off-targeting by TCR 47BE7 unlikely.These data support the notion that Hsf2 p.K72N 68-76 could behave as a "nonself" epitope, triggering a strong immune response not curtailed by immune tolerance.To understand the molecular mechanism of immunogenicity, we then determined the crystal structure of the ternary complex between TCR 47BE7 and H2-D b /Hsf2 p.K72N 68-76 .

Crystal structure of ternary complex
Given our structural data detailing the enhanced binding of the neoAg Hsf2 p.K72N 68-76 to H2-D b , we sought to understand the mechanism for its recognition by cognate TCR 47BE7.
We hypothesised that TCR recognition was mediated by the interactions between the TCR and mutant peptide-specific structural features of the pN 5 anchor and the solvent-exposed p6-p8 ridge.For these studies, we produced recombinant soluble TCR 47BE7 as previously described 56 .Correct folding and preserved substrate recognition in solution were determined by measuring binding kinetics between TCR 47BE7 and H2-D b /Hsf2 p.K72N 68-76 by biolayer interferometry (BLI).The 47BE7 TCR binds immobilised H2-D b / Hsf2p.K72N 68-76 with a high affinity typical for non-self-reactive TCR (K D 2.7 ± 0.3 µM) (Fig. 6a) 57 .The on and off-rates for complex binding were too fast to be calculated with precision.To determine the structural basis for epitope recognition and T cell activation, we crystallised and solved the TCR-47BE7/H2-D b /Hsf2 p.K72N 68-76 ternary complex structure to a resolution of 2.5 Å (Fig. 6b, Supplementary Table 2).Our data show that bound Hsf2 p.K72N 68-76 peptide was well defined on the electron density map, adopting nearly the same conformations in the binary and ternary complexes, respectively (0.6 Å Cα RMSD, Fig. 5b and Supplementary Fig. 9a).The well-defined electron density at the TCR:pMHC interface allowed for unequivocal placement of all critical amino acid side chains at the interface (Supplementary Fig. 9b).47BE7 exhibited conventional oblique docking geometry (13.13°incident angle, 57.49°docking angle) 58 .Notably, the TCR centroid was biased towards the peptide C-terminus, spanning the p6-p8 core segment identified in the binary structure and predicted to contribute to TCR binding.Binding between the TCR and the Hsf2 p.K72N 68-76 peptide was mediated by the C-terminal half of epitope comprised of pR 4 -[X]-pV 6 -pV 7 -pH 8 .In agreement, a comparison of solvent-exposed surface area (SASA) of the bound and unbound pMHC complex shows significant SASA reduction for pR 4 and pV 6 -pH 8 , which comprises the peptide contribution to the core epitope buried at the TCR-pMHC interface (Fig. 6c).For the remaining peptide residuesincluding the mutated residue pN 5 -SASA did not change significantly on complexation, indicating that these side chains remain buried or otherwise do not contribute to ternary complex formation.The buried residue H2-D b Y 156 projected into the E pocket in the pMHC structure was observed to rotate towards the D pocket in the bound structure, and the space previously occupied by Y 156 is instead filled with glycerol (Supplementary Fig. 9d, Fig. 6a).The presence of a glycerol contaminant in this position did not significantly alter conformations of other amino acid side chains, including the p6-p8 arched peptide conformation, and had no appreciable impact on the TCR-pMHC interface, but only reflected the flexibility of H2-D b Y 156 .The mobility of Y 156 observed in these structures could expand the size of the E pocket (Supplementary Fig. 9a-c), so it may accommodate the aromatic side chains of epitope residues at p5 or p6 including phenylalanine or even tryptophan 50 (Supplementary Fig. 9d, e, Supplementary Fig. 12).
The total area buried upon complexation between pMHC and TCR (1506 Å 2 ) was almost evenly divided between the two components (Fig. 6d, e).Interfacial contacts between 47BE7 and H2-D b /Hsf2 p.K72N 68-76 were mediated by complementarity determining region (CDR) loops CDR1α, CDR2α, CDR3α, CDR2β, and CDR3β, with little contribution from CDR1β (Fig. 6d, g, f).Binding of the TCR to H2-D b was mediated by the solvent-exposed residues Q 72 , R 75 , R 79 (H2-D b α 1 helix), as well as E 18 (H2-D b loop A), which undergo re-organisation upon complexation (Fig. 5g and Supplementary Fig. 9e-g), placing E 18 within the hydrogen bond distance of R 75 and R 79 and positioning R 75 to form hydrogen bonds with the hydroxyl and carboxyl groups of CDR2β S 51 and the Y 52 backbone carboxyl, respectively (Supplementary Fig. 9f).Additional interfacial TCR-pMHC polar interactions include hydrogen bonds between CDR2β M 56 and Q 72 , CDR3β E 97 and N 80 , as well as salt bridges between CDR3β E 97 and K 146 (H2-D b α2 helix).Polar interactions between TCRα and H2-D b were limited to a single hydrogen bond between CDR1α Y 32 and S 150 (H2-D b α2 helix) (Supplementary Fig. 9g).
The polar interactions between the peptide and TCR involved the CDR3α Y 94 hydroxyl and the pR 4 backbone carboxyl; the CDR3β Y 101 hydroxyl and the pH 8 side chain imidazole and backbone amide; as well as a salt bridge between CDR3β E 97 and pH 8 .Other electrostatic interactions were observed in the form of water bridging between CDR2β D 57 as well as CDR3α N 95 and pR 4 .Finally, non-polar van der Waals (VDW) contacts were between CDR1α Q 31 and Y 32 and pV 6 and, as well as between CDR3α Y 32 , CDR3β Y 101 and pVal 7 (Fig. 5h, i).Collectively, our data illustrate that the TCR 47BE7epitope interaction is biased towards the peptide C-terminus, in a region of pre-existing structural rigidity within H2-D b /Hsf2 p.K72N 68-76 binary complex.While the mutant pN 5 residue lies within the core pR 4 -[X]-pV 6 -pV 7 -pH 8 epitope, it makes no significant side-chain contacts with TCR 47BE7, confirming its minimal direct impact on TCR binding.The structure of TCR 47BE7:H2-D b /Hsf2p.K72N shares common features with published TCR-H2-D b complexes found in the PDB and TCR3D databases (n = 16) 58 .Despite structural similarities, each TCR adopts a distinct orientation with respect to cognate binary complex, with interface area or the TCR docking angle values largely varying, with the most similarity observed between the 47BE7 structure (7NA5) and (7N4K) (Supplementary Fig. 10a, b).The conformation of each binary complex was minimally affected by the nature of the bound epitope, where each peptide formed a typical p6-p8 C terminal arch (Supplementary Fig. 10c).The larger docking angle value inversely correlated with smaller interface area (Supplementary Fig. 10b), but the latter usually does not correlate with TCR avidity or affinity 59 .Instead, the smaller buried interface in the 47BE7 structure supports a significant contribution from the bound epitope, potentially increasing TCR selectivity.
The complex between the WT peptide and H2-D b was unstable and not available for further analysis.Consequently, the molecular basis for discrimination between the WT and mutant Hsf2 68-76 peptides remains unclear, although a few plausible scenarios exist, corroborated by the available biochemical data and molecular modelling.Our study indicated that the WT peptide has low affinity towards H2-D b (Fig. 5a).Other pK 5 peptides follow a similar trend.For instance, the affinity for pN 5 peptides was significantly higher as compared with the non-pN 5 peptides, whereas all the pK 5 peptides displayed low affinity with IC 50 > 1 μM (Supplementary Fig. 11a).It is noteworthy, that among 2276 H2-D b -specific 9-amino acid (AA) peptides representing mouse immunopeptidome, only 3 (~0.1%)had lysine residue at p 5 .Further, the WT Hsf2 68-76 epitope was not detected in any mouse tissue 55 .
To determine the structural basis for discrimination between pK 5 and pN 5 peptides, we performed in silico docking of WT Hsf2 68-76 using PepFlexDoc and the crystal structures we solved as templates.In total we have utilised the following models: (N5) H2-D b structure with the MT epitope as a benchmark, (K5a) H2-D b structure in which pN 5 was replaced with lysine in anchor orientation, (K5na) H2-D b structure in which pN 5 was replaced with lysine in a non-anchor orientation, and (K5a_Y156a) H2-D b structure in which pN 5 was replaced with lysine in anchor orientation and with Y 156 adopting the alternate conformation observed in the ternary complex.Each model was reduced to the H2-D b domain 1 (AA residues 1-180) and peptide ligand.The top 10 docking scores from each of these scenarios are plotted, with the N5 scenario scoring the highest (Supplementary Fig. 11b).Other scenarios produced more widespread and overlapping solutions, with K5a-Y156a being the least plausible.Comparison of the top 10 solutions from scenarios K5a and K5na represent the two alternate conformations for the H2-D b /Hsf2 68-76 complex.The pK 5 non-anchor orientation (K5na) resulted in docking solutions with WT peptide conformations distinct from that of MT epitope (Supplementary Fig. 11c, d), whereas the H2-D b residues retained their original conformations.By contrast, the pK 5 anchor orientation (K5a) resulted in the WT peptide conformations similar to that of MT epitope found in the crystal structure.However, to accommodate the pK 5 side chain inside the binding pocket and to avoid inter-atomic clashes, the bulky aromatic side chain of H2-D b W73 required drastic conformational change (Supplementary Fig. 11e, f).Based on the docking scores, both scenarios remain possible, but probable modelling bias must be accounted for.The body of evidence indirectly favours the non-anchor pK 5 conformation, as rotation of a solvent-buried tryptophan side chain toward exposed orientation seems unlikely and has not been previously been observed in H2-D b .Moreover, HLA or H2 molecule promiscuity is largely the result of peptide structural plasticity combined with limited specificity, defined as peptide binding motifs 59 .In any case, the modelling and docking experiments clearly indicate that the H2-D b C pocket is unable to accommodate the extended and basic pK 5 side chain without structural re-arrangements eliminating steric clashes between peptide and the H2-D b residues lining the peptide binding site.Thus, binding of WT Hsf2 68-76 may yield either a distinct peptide conformation or distortion of the H2-D b binding pocket structure.In such scenarios, the interface between H2-D b /WT Hsf2 68-76 and TCR 47BE7 would be severely compromised due to clashes between either exposed pK 5 (model K5na) or between the exposed H2-D b W73 side chain (model K5a) from one side and TCR residues from another.
Collectively, we determined the molecular mechanism of immunogenicity for the H2-D b -restricted B16F10 melanoma neoAg Hsf2 p.K72N 68-76 .This neoepitope is discriminated from the WT peptide at the MHC level due to a K72N amino acid substitution at the anchor position p5 and can be classified as a group II neoAg 27 .At the same time, it is discriminated at the TCR level, as the H2-D b / Hsf2 p.K72N 68-76 complex adopts a unique conformation specifically recognised by TCR 47BE7, whereas the WT peptide is not efficiently recognised.In addition, the earlier published data show an apparent lack of epitopes cross-reactive to Hsf2 p.K72N 68-76 across the mouse immunopeptidome.Based on all of the above, we conclude that the neoAg Hsf2 p.K72N 68-76 is a unique "non-self", prototypical neoantigen in B16F10 melanoma.

Discussion
Neoantigens (neoAg) have garnered significant interest as therapeutic targets due to their potential to exhibit enhanced therapeutic efficacy and safety.While studies have described features of immunogenic neoantigens based on post hoc computational analysis 60 , detailed molecular and structural studies of neoAg and corresponding TCR remain limited.Our study aimed to address this by investigating fundamental assumptions regarding the molecular and structural determinants of neoantigen immunogenicity.We did so in the B16F10, a preclinical model with a well-characterized response to existing immunotherapies and, importantly, with other known antigens available for future comparative studies.Previously, many of the B16F10 antigens studied were either tumour-associated antigens (typically melanocyte differentiation antigens such as gp100 or Trp2) [40][41][42] or OVA 257-264 , a foreign antigen engineered to be artificially expressed by B16F10 32 .Others have investigated neoantigens in B16F10, uncovering immunogenic epitopes (distinct from those discovered in our study) that elicit tumour growth reduction upon peptide vaccination 61 ; however, extensive analyses of neoAg-reactive TCRs in B16F10 and their structure-function relationships were not performed previously.
We first identified and characterised several TCR-antigen pairs from the widely-utilised B16F10 melanoma model.We then completed in-depth biochemical and structural studies of the prototype anchor residue-modified neoantigen H2-D b /Hsf2 p.K72N and the corresponding monoclonal TCR 47BE7.While H2-D b /Hsf2 p.K72N has been identified in a previous analysis of B16F10 60 , we were able to demonstrate its immunogenicity, identify an Hsf2-reactive TCR, and conduct its comprehensive structure-activity relationship (SAR) characterization.We selected H2-D b /Hsf2 p.K72N 68-76 and 47BE7 for characterization due to demonstrable in vitro and in vivo activity in a challenging tumour model.Importantly, while vaccination with Hsf2 p.K72N 68-76 peptide suppressed tumour growth, ACT shows significant anti-tumour effect only with Hsf2 p.K72N 68-76 overexpressed by B16F10 cells.This divergence indicates that the intrinsic level of neoAg expression is not sufficient for tumour suppression using ACT alone.This observation is in accordance with published data demonstrating that antigen-transduced (OVA, Pmel-1) B16F10 is highly resistant to ACT and often requires combinatorial strategies (i.e., checkpoint blockade) and/or overexpression or peptide/TCR modifications to impart efficacy 33,34,62 .For the purposes of this study, vaccination and ACT serve as proof-of-concept that various immunotherapeutic modalities involving Hsf2 p.K72N can yield tumour growth control in vivo.Given that 47BE7 exhibited high functional avidity and limited cross-reactivity in our in vitro studies, we hypothesised this model could provide insight into mechanisms of B16F10 resistance to immunotherapy.
We found that the lysine to asparagine (p.K72N) anchor residue substitution results in a 175-fold improvement in the surface presentation of H2-D b /Hsf2 p.K72N 68-76 compared to that of the wildtype Hsf2 68-76 .The crystal structure of H2-D b /Hsf2 p.K72N 68-76  demonstrates that the mutated pN 5 residue is directly responsible for this effect, due to stabilizing polar interactions between the Hsf2 p.K72N epitope and H2-D b Q 97 .This anchor residue mutation also induced the formation of a rigid, solvent-exposed, hydrophobic arch at the carboxy-terminal pV 6 -H 8 segment of the Hsf2 p.K72N 68-76 , which was essential for binding to TCR 47BE7.The structural stability of neoAg pMHC has been repeatedly associated with immunogenicity, often attributed to increased pMHC surface abundance due to slow peptide dissociation kinetics 45 .Our data reaffirm and extend this finding, showing that stability measures may indirectly capture fine structural features within an epitope that contribute to immunogenicity through TCR contacts, exemplified by the rigid hydrophobic pV 6 -pH 8 arch.
Recently, several groups have published structural studies of human neoAg-reactive TCR [27][28][29][30]63,64 . The sructural data we present support and expand on these earlier findings in several noteworthy ways.First, we observed high-level commonalities between TCR 47BE7/Hsf2 p.K72N and TCR9a/TCR10, as well as TCR4, which bind to the group II (anchor-residue modified) neoAg HLA-C*08:02/Kras p.G12D 27 These TCRs employ a similar binding mode characterized by multiple intermolecular contacts distributed across the TCR:pMHC interface.As in TCR 47BE7, experimental modification of contact residues within the core TCR epitope eliminates TCR reactivity, suggesting that the totality of the interface is necessary for TCR binding 27 .This binding mode often contrasts with that employed by neoAg-reactive TCR that bind to group I (solventexposed residue modified) neoAg such as TCR12-6/TCR38-10, which bind to the neoAg HLA-A*02:01/TP53 p.R175H 29 .In this latter circumstance, the observed TCR contacts are biased towards the solvent-exposed mutant residue and avoid contacts with the remaining peptide surface.While we did not perform structural characterization of our 29BF8 TCR (recognizing H2-K b Lrc28 p.K329T) or our 44CH2 TCR (recognizing H2D b Pbk p.V145D), we would hypothesise that they employ a similar binding mechanism to Group I neoAgs.Further elucidation of the Group I and Group II neoAgs and their respective TCRs may further guide identification of effective neoAg-specific TCRs in the future.
We acknowledge several limitations of our study, primarily being restricted to a murine model system, which limits direct translation to the human context.We also focused on a single neoantigen-TCR pair, in the context of a single MHC-I allele, and while we note that ~40% of H2-D and H2-L alleles share similar structural features, this possibly limits generalizability outside of this context.However, we have created a model system of neoAgs in a commonly used preclinical model; this may enable study of low-vs high-affinity neoAgs and cross-reactive vs mutant peptide-specific TCRs alike, thus enabling us to create an atlas of neoAg pMHC-TCR interactions and their relationship to immunotherapy efficacy.More specifically, our finding that our highest affinity TCR, 47BE7, but not other neoAg-reactive TCRs, can recognise tumours and control tumour growth confirms the importance of high affinity TCRs in enabling recognition of neoAgs, which despite their high theoretical immunogenicity, are often expressed at a lower level than TAAs in tumours 65 .Future studies should perhaps consider selecting high avidity TCRs that enable potent anti-tumour immune responses, provided that these TCRs are not cross-reactive with healthy tissue or cause signalling fatigue, as has been previously observed [66][67][68] .Further, we have identified a neoantigenic structural motif in the context of H2-D b , which supports strong pMHC-TCR interactions that mediate robust complex stability and is the basis of 47BE7's high affinity for its cognate pMHC.While this may seem unique to our model, as described above, we observe commonalities with human TCRs previously studied and provide evidence that TCRs exhibiting similar features to ours will be good candidates for vaccination with pertinent neoAg peptides and perhaps for adoptive cellular therapies.Further, using the SAR approach, we fully validated the group II neoAg Hsf2 p.K72N 68-76 and determined the mechanism of its immunogenicity at the molecular level.Altogether, we have identified neoAgs and neoAg-reactive T cells in B16F10 and have provided functional and structural data on a high affinity TCR, 47BE7, that confers tumour protection and could inform the study and identification of human neoAg-reactive TCR biology in the future.

Methods
All research complies with ethical regulations.Mouse procedures and monitoring protocols were approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC) protocol: 15-2171, approval: IACUC-2016-0028.
Whole exome sequencing B16F10 (tumour) cells were expanded in culture to 75% confluence.Total splenocytes (germline) were isolated from a male C57BL/6 colony founder.Genomic DNA was isolated using DNeasy Blood & Tissue kit (Qiagen, cat#: 6950).Whole exome sequencing (WES) libraries were prepared using SureSelectXT Mouse All Exon kits (Agilent, cat# G7550A).Paired-end 100 bp sequencing was performed using HiSeq2500 reagent kit v3 (Illumina, CA) targeted sequencing depths of 300x and 150x for tumour and germline samples, respectively.Sequencing reads were mapped to GRCm38.p6/mm10 using BWA-MEM 69 .Duplicate read marking and base quality score recalibration were performed using GATK/Picard 70 .Somatic variant calling was performed for target regions using MuTect and Strelka with default filters 71 .
Isolation of tumour mRNA and RNA sequencing B16F10 cell tumour cells (1 × 10 6 ) were inoculated into the dermis of subject animals.Seven days post-inoculation the tumours were resected, and total RNA was isolated using RNeasy kits (Qiagen, cat#: 74104).Messenger RNA sequencing library generation was performed using Ribo-zero magnetic gold and TruSeq RNA Sample preparation kits (Illumina, CA).Paired-end, 100 bp, sequencing was performed using a HiSeq 2500 reagent v3 kit, with a targeted sequencing depth of 1 × 10 8 reads/library.Sequencing reads were mapped to GRCm38.p6/mm10 using HiSa.

Identification of mutation-derived tumour neoantigens (neoAg)
Mutation-derived tumour neoantigens were identified using our established pipeline 36 .Briefly, somatic variants are identified by WES.Variant expression is quantified by local assembly and allele-specific quantification of mutated and reference transcripts.Variant transcripts are translated in silico.The peptide-MHC-I binding prediction tool NetMHCpan (v.4.1) was then used to identify candidate neoAg for further study.Code is available at https://github.com/openvax/vaxrank.

Peptide synthesis
Experimental peptides were individually custom synthesised via the solid-phase method and validated by GenScript (Piscataway, NJ), with standard removal of trifluoracetic acid and replacement with hydrochloride, purified to >98% by HPLC, and lyophilised for storage.Peptides were reconstituted in DMSO at 10 µM and frozen at −80 °C until use.

Immunization
Peptide-based vaccines comprising peptide antigen and TLR7/8a adjuvant co-delivered in self-assembling particles (referred to as "SNAPvax TM ") were produced in accordance with standard protocol 38,39 .Briefly, peptide antigens were synthesised and were linked to imidazoquinoline-based TLR7/8a (Barinthus Therapeutics, USA) using an azide-alkyne cycloaddition click chemistry reaction.Vaccines were reconstituted in sterile phosphate-buffered saline to a final concentration of 40 µM, and 50 µL was injected subcutaneously to bilateral footpads.
8-12-week-old animals balanced and completely randomised with respect to age and sex, were used for all immunization, adoptive cell transfer, and tumour allograft experiments.Subjects were evaluated every 48 h for the full duration of all experiments.Euthanasia was performed by carbon dioxide asphyxiation followed by cervical dislocation.
Peripheral blood was obtained by submandibular vein puncture.Approximately, 250 µL of was collected into sterile heparinised tubes.Red blood cells were removed with ammonium-chloridepotassium (ACK) osmotic lysis solution; 2:1 v/v for 5 min, followed by centrifugation at 500 × g, 5 min.The resulting peripheral blood mononuclear cells (PBMC) were washed twice with MACS buffer (PBS, BSA 2%v/v, EDTA 2 mM), and then stored in MACS buffer at 4 °C until use.
Total splenocytes were obtained by splenectomy.Tissue samples were macerated over 70 µm pore-size nylon filters.Red blood cells were removed by treating the samples with ACK lysis solution; 2:1 v/v for 5 min, followed by centrifugation at 500 × g, 5 min.Total splenocytes were washed twice with MACS buffer (PBS, BSA 2%v/v, EDTA 2 mM) and maintained at 4 °C until use.

Tumour inoculation
Orthotopic injections of B16F10 melanoma cells were performed 72,73 .Briefly, subjects were sedated and paralysed by administration of 1:2:7 %v/v xylazine: Ketamine: deionised water, 100 µL, intraperitoneal (IP) injection, once.Mechanical shears were used to remove hair from a 2 cm 2 body surface area overlying the posterior hindlimb.Sterile 70% v/v Ethanol: deionised water swabs were used to remove disinfect the skin surface.Tumour cell suspensions containing 100,000 cells in 100 µL were loaded into syringes with permanent 28 G needles.Needles were inserted into the skin to the level of the dermis and the full volume was injected.Subjects were returned to the enclosure and monitored for complete recovery from anaesthesia.Humane endpoints for subject withdrawal were as follows: tumour diameter ≥15 mm, ulceration or body condition score <3 (ordinal scale).

Lentivirus plasmids for creation of tumour antigenoverexpressing tumour lines
Antigen-overexpressing tumour lines were engineered to normalise antigen levels; notably, Hsf2 is expressed at lower levels than nonmutated tumour antigens studied 81 .pENTR Gateway DONOR plasmids were constructed as follows.pEF-ENTR-A (696-6) was linearised with BamHI (NEB, Cat#: R3136S) and EcoRI-HF (NEB, Cat#: R3101S).The cDNA for mTagBFP2; G/Gly-S/Ser-G/Gly spacer; T2A; followed by the cDNA corresponding to the 25 amino acid segments (Supplementary Table 3) surrounding indicated neoantigen peptides and a c-terminal flag (-DYKDDDDK) tag were synthesised (Genscript, NJ).Segments were assembled in series into the linearised pENTR-A backbone using the flanking homology method (NEBuilder HiFi DNA Assembly, NEB).pLenti transfer vectors were generated using LR Clonase II (Thermo-Fisher, Cat#: 11791020).Sanger sequencing (Genscript, NJ) was used to verify the correct sequence, order, and orientation of all constructed plasmids.
Expression, folding and purification of H2-D b /YGFRNVVHI Soluble peptide-MHC monomers were synthesised in accordance with published methods 85 .Accordingly, the extracellular domain of H2-K b , H2-D b , as well as full-length human β2M (hβ2M) were cloned into pET3A plasmids (provided by Ton Schumacher, Netherlands Cancer Institute-Antoni van Leeuwenhoek (NKI-AVL)).For MHC tetramer production H2-K b and H2-D b expression constructs included a c-terminal biotin acceptor peptide (BirA-tag).Proteins were expressed in E. coli BL21(DE3) PLysS as inclusions bodies (IBs).IBs were solubilised in 100 mM Tris-HCl, pH 8.0, supplemented with urea 8 M, DTT 100 µM, and EDTA 10 mM.Insoluble material was cleared by centrifugation 20,000 × g, 20 min.Denatured proteins were added in a H2-D b :hβ2M 1:2 mol:mol ratio to solution of Tris-HCl 100 mM pH 8.0, supplemented with L-arginine 400 mM, L-glutathione 500 µM, oxidised L-glutathione 50 µM, and EDTA 2 mM, PMSF 10 mM, protease inhibitor cocktail (Roche, Cat#: 11873580001) and peptide 1.2 mM.The mixture was stirred at 4 °C for 72 h, and precipitate cleared by centrifugation at 20,000 × g, x 10 min, concentrated by centrifugal ultrafiltration using a 15 kDa membrane (Millipore, cat#: UFC903024) and sequentially purified by size exclusion chromatography (Superdex 75, GE Healthcare), followed by anion exchange (HiTrap Q HP 5, GE Healthcare).MHC monomers were concentrated by centrifugal ultrafiltration using a 4 kDa membrane (Millipore, cat#: UFC8030) and exchanged into HBS (HEPES 10 mM, NaCl 150 mM).Peptide-MHC protein complex identity was verified by polyacrylamide gel electrophoresis, then split into single-use aliquots before snap-freezing in liquid nitrogen, then stored at −80 °C until use.For MHC tetramer production site-specific enzymatic biotinylation by BirA biotin ligase was performed before the anion-exchange chromatography previously described.

Biolayer interferometry
The AVI-tagged version 86 of recombinant H2-D b was used to refold the pMHC in complex with YGFRNVVHI peptide; this was biotinylated using a BirA biotinylation kit (Avidity) and purified by gel filtration.The gel-shift assay using streptavidin (Fisher Scientific) verified protein biotinylation both after biotinylation and immediately before starting the BLI experiment.
Binding between pMHC and TCR was measured using a BLI Octet RED96 instrument and streptavidin-conjugated SA chips in accordance with the manufacturer's (Pall ForteBio, Menlo Park, CA) protocol.For all analyses, HST buffer (10 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1% BSA) was used.Biotinylated pMHC was diluted to 2-10 μg/mL, immobilised on a sensor chip, and equilibrated in HST buffer for 1 min.Following this step was TCR association (60 s) in 200 µL of TCR solution (concentrations ranging between 0 μM and 20 μM), and then dissociation in 200 µL of HST buffer for 3-20 min.Assays were conducted at 25 °C.The Octet® 9.1 System Data Analysis software was employed to determine dissociation constant values from these data.

Protein crystallization and data collection
TCR 47BE7 and H2-D b / 68 YGFRNVVHI were refolded and purified separately as described above.Protein crystallization was performed by sitting drop vapour diffusion.96-well INTELLI-plates (Art Robbins Instruments, Cat# 102-0001-20) were seeded with a Mosquito crystallization Robot (SPT Labtech) utilizing a 1:1 v/v protein to precipitant ratio then incubated at 18 °C until crystal formation.H2-D b / YGFRNVVHI formed prism-shaped crystals (Tris-HCl 0.1 M, pH 8.5, sodium acetate 0.2 M, PEG3350 20-25%).Crystals were cryoprotected with the same mother liquor, supplemented with ethylene glycol 25% v/v then flash-frozen in liquid nitrogen and stored until data collection.
X-ray diffraction data collection, structure solution and refinement X-ray diffraction data were collected at Argonne APS beamline 19BM using SBCCollect.Data were indexed, integrated, and scaled using HKL3000 and the AIMLESS/CCP4 programme suite 87 The crystal structure of H2-D b /YGFRNVVHI was solved using PHASER with the reference search model (PDB: 5OPI) and refined using REFMAC and COOT 0.9.7 88 .The H2-D b /YGFRNVVHI structure coordinates and structure factors are accessible via Protein Data Bank accession code (PDB: 7N9J).The structure of TCR 47BE7/H2-D b / 68 YGFRNVVHI ternary complex was solved using the binary complex as a search model, the 2FoFc map of which was used to sequentially build and refine the TCRα and TCRβ chains for TCR_47BE7 heterodimer by alternating refinement in REFMAC with model building and refinement in COOT.The 47BE7/H-2-Db/ 68 YGFRNVVHI ternary complex coordinates and structure factors are accessible via Protein Data Bank accession code (PDB: 7NA5).The X-ray diffraction data collection, processing and refinement statistics are presented in Supplementary Table 2.
Solvent exposed surface area (SASA, A 2 ) for individual amino acid residues was calculated using NACCESS software 89 , and was expressed as a percentage of all residue surface area (ASA).The SigmaA-weighted 2Fo-Fc or Fo-Fc electron density maps in CCP4 format for drawing were generated in COOT 0.9.7 using amplitude (FWT) and phase (PHWT) data from the REFMAC mtz output structure factor file.

Structural modelling
Structural modelling of wild-type H2-D b /Hsf2 68-76 was performed using PepFlexDock in Rosetta.The high-resolution crystal structure of H2-D b /Hsf2 p.K72N 68-76 served as the template, into which the position 5 asparagine to lysine mutation was introduced using COOT or the PyMol mutagenesis function.Energy minimization was then performed using FlexPepDock online server 90 .Each model was truncated to the H2-D b domain 1 (AA 1-180) and peptide.The top scoring models were compared in COOT, PyMol.Images were generated using PyMol and Microsoft Excel 2019.

Fig. 1 |
Fig. 1 | Identification of neoantigenic epitopes in the B16F10 melanoma model.a Schematic (made using Biorender with full license) depicting B16F10 neoantigen identification.b C57BL/6 mice (n = 5 independent biologic replicates) were immunised with peptide vaccine targeting putative B16F10 neoantigens.Seven days post-immunization splenocyte-derived T cells were stimulated with mutant peptide (solubilised in DMSO) for 6 h then IFNγ production was measured by flow cytometry.Symbol indicates individual mice (n = 5/condition), error bars indicate the group median, ±95% confidence interval (CI).Solid line indicates assay lower limit of detection (LLD).Dashed indicates upper limit of 95% CI for negative responses.Statistical analysis: one-way Kruskal-Wallis test, followed by Dunn's test for multiple comparisons, with alpha level set to 0.05.Colour key: red; T cellelicited response significantly above the LLD, grey; insignificant T cell response, black; T cells not stimulated with any peptide (DMSO only control).Where p < 0.001, p value was too low for Prism software to provide an exact value.c C57BL/6 mice (n = 4/group, repeated four times) were immunised with (long) peptide vaccine targeting putative B16F10 neoantigens.Seven (7) days post-immunization, flow cytometry was performed on splenocyte-derived CD8 + T cells.Flow cytometry plots are organized in columns; data from vaccinated mice (left) vs unvaccinated (right).Representative tetramer staining is shown.Gating strategy shown in Supplementary Fig. 2. d As in (b).Error bars depict the group median, ±95% confidence interval (CI); n = 5 independent biologic replicates.Peptide stimulation of T cells by tumour-associated antigens (TAAs) [blue], (non-tumour) OVA antigen [dark grey], neoantigens eliciting measurable tetramer response [red], neoantigens not eliciting measurable tetramer response [light grey].e Attributes of neoantigens (top) predicted computationally and TAAs previously characterised 40-42 .Binding affinity (BA)-Rank values are shown for mutated (ALT) and wild type (REF) peptides.Table subscript legend is as follows: 1 NetMHCpan v.4.1 BA-Rank% value. 2 Variant a.a.position vis-à-vis learned FlexPepDock structure.f In silico MHC-I binding affinity analysis of Hsf2 p.K72N 68-76 demonstrates high affinity binding and high differential binding affinity between ALT and REF peptides, hereafter referred to as mutant (MT) and wild type (WT) peptides, respectively.Source data are provided as a Source Data file.

Fig. 2 |
Fig. 2 | Neoantigen-reactive CD8 + T cells recognise cognate peptide in vitro.a Table detailing attributes of tumour-reactive T cell receptors (TCRs), including neoantigen-reactive (top) and tumour-associated antigen-reactive (TAA-reactive) (bottom) TCRs isolated upon vaccination.b Neoantigen-reactive CD8 + T cell responses were induced by vaccination; isolated tetramer + T cells were stimulated for 6 h with (varying concentrations of) cognate peptide and αCD28 and subsequently analysed via intracellular staining (ICS) flow cytometry for IFNγ expression.c Transgenic (tg)TCR CD8 + T cells were co-incubated with varying concentrations of cognate mutant (MT) peptide or wild type (WT) peptide (x-axis) and αCD28 for 6 h; intracellular staining (ICS) was performed subsequently.Percentage of T cells expressing IFNγ of a parent CD3 + CD8 + population is shown on y-axis, normalised to maximum IFNγ expression.MT peptide values are shown as clear circles, and WT as filled, black circles.Trp2-reactive TCR 180CC6 only recognises a WT peptide (shown with clear circle).d Neoantigen-reactive TCR half-maximal (EC 50 ) cytokine production concentration (top).TgTCR CD8 + T cells expressing the indicated TCR were stimulated, as described in (c), with titrated mutant (MT) or wild-type (WT) peptide and IFNγ production was measured by ICS.Symbols indicate median of biologic replicates (n = 3/condition), ±95% CI.Dashed horizontal line indicates mean half-maximal response (EC 50 ) for tested neoAg TCR.(Bottom) Ratio of WT/ MT EC 50 as log 10 fold change.Source data are provided as a Source Data file.

Fig. 4 |
Fig. 4 | Hsf2 neoantigen-reactive CD8 + T cells elicit anti-tumour activity in vivo.a C57BL/6 mice (n = 16 independent biological replicates in control group and 26 independent biological replicates in the vaccinated group) were treated with Hsf2 neoantigen minimal epitope (YGFRNVVHI) vaccine or PBS (mock).Tumours were monitored via calipers.b Tetramer staining on CD3 + CD8 + T cells isolated from tumour-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMCs) from mice vaccinated with each of the neoantigens listed, as described in 2b.Symbol indicates individual mice, error bars depict the group median, ±95% confidence interval (CI) ± 95% confidence interval (CI), n values (shown on figure) represent independent biological replicates.Statistical analysis consisted of twosided unpaired t-test, followed by Benjamini, Kreiger and Yekutieli two-stage step-up method, with desired false discovery rate (Q) of 5.00%.Ndufs6: p = 0.0000000279, Nsun2: p = 0.0000000825, Hsf2: p = 0.00053.c Schematic (made using Biorender with a full license) describing administration of adoptive cell transfer (ACT) of Hsf2-reactive T cells in vivo.n = 15 (vehicle group), 14 (OT-1 group), or 11 (Hsf2 group) C57BL/6 mice (independent biological replicates) collected over 3 independent experiments.d tumour growth was measured over time by calipers every 2-3 days and plotted using Graph Pad Prism 7. Error bars depict standard error of the mean.**p = 0.0036 (vehicle vs. Hsf2-reactive T cell ACT comparison), while for the comparison between OT-1-reactive T cell ACT and Hsf2reactive T cell ACT, p = 0.0122.Two-way ANOVA with Tukey correction.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | Structure of the pMHC binary complex.In selected figures, one alternate conformation for pR 4 was excluded for clarity.a RMA-S cells were plated at 25 °C for 18 h, then co-incubated with the indicated peptides for 30 min at 30 °C, followed by 3 h at 37 °C.Surface H2-D b geometric mean fluorescence intensity (gMFI) was measured by flow cytometry and plotted for peptide concentrations tested.Symbol indicates the mean of biologic replicates (n = 3), ±95% confidence interval (CI).b Crystal structure of H2-D b /Hsf2 p.K72N 68-76 .H2-D b is coloured in grey, human β2M (hβ2M) is shown in blue.pN 5 refers to the asparagine (N) located at p5. Peptide stick model: oxygen coloured red, nitrogen is blue and carbon is grey.SigmaAweighted Fo-Fc electron density map (δ = 4.0, radius = 1.5 Å) is shown around peptide.c The peptide binding domain of H2-D b (cartoon) and Hsf2 p.K72N 68-76 are coloured according to B-factor values.Peptide residues shown as sticks.The SigmaA-weighted 2Fo-Fc map (σ = 1.0, radius = 1.5 Å) is superposed onto peptide residues only.d Peptide-binding cavity of H2-D b is shown as a surface area coloured according to the surface charge.Hsf2 p.K72N 68-76 residues are shown as sticks, with atoms coloured according to charge.The approximate location of each binding pocket is marked by a letter from A (N-terminal pocket, residue P1) to F (C-terminal pocket, residue P9).Topamino acid sequences for mutant (MT) and wild type (WT) Hsf2 peptide, anchor and buried residues (SASA < 20%) are depicted in bold.e Arrangement of residues in the C-pocket of the binary complex.Cartoon and stick model.Hsf2 p.K72N 68-76 carbon atoms are coloured in yellow, H2-D b carbons are coloured in grey.Oxygen atoms are red, nitrogen, blue.H-bonds are dotted lines.f Peptide scan.CD8 + TCR-47BE7 + T cells were incubated with 1 μM of each YGFRNVVHI peptide variant from a positional scanning library, along with αCD28, for 6 h and IFNγ production measured by intracellular flow cytometry staining (gating strategy shown in Supplementary Fig. 8).Colour indicates mean of biologic replicates (n = 2).Boxed squares indicate native amino acid at indicated position within mutant peptide YGFRNVVHI.Source data are provided as a Source Data file.

Fig. 6 |
Fig. 6 | Structure of the pMHC-TCR ternary complex.a Kinetics of soluble TCR-47BE7 binding to immobilised H2-D b /Hsf2 p.K72N as determined by biolayer interferometry.The dissociation constant (K D ) was determined by curve fitting in Octet® 9.1 System Data Analysis software.b Structural superimposition of TCR-47BE7/H2-D b /Hsf2 p.K72N 68-76 (7NA5) and H2-D b /Hsf2 p.K72N 68-76 (7N9J) structures.(Inset) Superposition of the corresponding Hsf2 p.K72N 68-76 peptides.c Residue-specific solvent-accessible surface area (SASA) in unbound/binary (grey) and bound/ternary (blue) structures were calculated using NACCES 89 .d TCR footprint on pMHC (inter-atomic distance cutoff 4 Å).En face view of H2-D b /Hsf2 p.K72N 68-76 with superimposed TCR-47BE7 CDR loops with residues as sticks.H2-D b interface is coloured according to observed CDR1ɑ-CDR3ɑ (orange), CDR1β-CDR3β (red), or no TCR contact (white).Peptide interface is coloured blue.e TCR-pMHC interface distribution between components.f Distribution of inter-atomic interface contacts between TCR and pMHC, distance cutoff 8 Å. Computed by NACCESS, as described in Methods.g Interface H2-D b (green) and peptide (blue) residues, stick models.Inter-atom distance cutoff 4 Å.h The interface between bound neoepitope and TCR-47BE7 in the pMHC-TCR structure in cartoon model.The polar bonds are depicted as dotted lines.Only the TCR residues that are in direct contact (<4 Å distance) with epitope atoms are shown as sticks.i Interface between Hsf2 p.K72N 68-76 (atoms presented as VDW spheres) and TCR-47BE7 (the protein surface was coloured according to the TCR chains).One alternate conformation for pR 4 was excluded for clarity.Source data are provided as a Source Data file.